Micro mechanical methods and systems for performing assays

ABSTRACT

The present invention provides a micro mechanical system for performing assays for determining the presence of one or more selected analytes in a sample. The device comprises of a base and a disposable strip with at least one reaction well and at least one moveable member capable of moving fluids and parts through the fluids in a defined reaction well. Reagents in the reaction chambers and or the moveable members, react with the sample to yield a physically detectable change. The moveable parts are capable of executing motions that either mix, move reaction components, exchange or systematically deliver reagents to targets in the cartridge. Sensors in the base are configured to detect and or quantify the presence of a sample in the reaction well and of analytes in the sample. The signal is converted to an output on a visual display window on the external part of the base.

RELATED APPLICATION DATA

This application claims the benefit of U.S. provisional patentapplication No. 60/515,731 filed on Oct. 29, 2003, which is hereinincorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates to microfluidics systems for performingassays for determining the presence of one or more selected analytes ina sample.

BACKGROUND

Qualitative and quantitative immuno- and chemical assays have gainedacceptance as important tools in the medical and food industries. Thesemethods have been used for the diagnosis of disease conditions,detection of analytes, and for the detection of microbes, such asbacteria. These methods of diagnosis have established effectiveness, andthe methods have made it easier for physicians to monitor and managepatients undergoing various forms of therapy.

Traditionally, the diagnostic assays have been performed in hospital andclinic settings, and involve the use of sophisticated and expensiveequipment, that require specially trained personnel for their operation.Further, the assay results are sometimes not available for days or weeksafter the samples from the patients have been obtained. The presentlyavailable diagnostic assays are thus costly, time consuming, and notconvenient.

Attempts have been made to develop less costly assays. For example, atypical home self-test for detecting blood components requires thepatient to prick a finger with a sterilized lancet, apply a drop ofblood sample to a sample application area on the disposable strip, andthen wait for the results. Assays that use other bodily fluids, such asurine essentially work in a similar manner. These devices are designedsuch that a typical lay person can perform the assays correctly withvery little training. However, these assay systems generally suffer fromlow accuracy or require a number of preparative steps be performed thatcould compromise the test results, and are thus not convenient.

U.S. Pat. No. 5,580,794 to Allen Michael describes a single useelectronic assay device that assays for specific analytes in a givensample. U.S. Pat. No. 4,806,312 to Greenquist describes a multizoneanalytical element having detectable signal concentration zone. U.S.Pat. No. 4,627,445 to Garcia et al. describes a hand-held portablemedical diagnostic system for checking measurement of blood glucose,urea nitrogen, hemoglobin, or blood components, where a disposableneedle or lance probe package carries a chemical reagent strip such asblood reacting chemistry, a visual readout, and a computer system.

U.S. Pat. No. 4,197,734 to A. Rosenberg describes an apparatus that iscapable of measuring the clotting time of blood. The apparatus includesa support frame, which supports a syringe containing a blood sample, anda turntable that rotates. Blood from the syringe drops onto theturntable where the clotting time is automatically and graphicallydepicted by a chart that is rotated on the turntable. The apparatus canalso be employed to determine variations in the viscosity of bloodplasma and other fluids.

U.S. Pat. No. 3,486,859 to Greiner et al. describes an apparatus thathas a double arm holder with blood liquid reactant chambers that areconnected to each other via a small capillary conduit. An air pump isprovided for applying pressure changes to one of the chambers to effectperiodic mixing of the liquids via the capillary conduit. Indicatormeans are included to detect the progressive restriction of thecapillary conduit upon coagulation of the blood.

The methods described above have severe limitations which make themextremely challenging for home use. Some of the methods require specialblood preparations and handling, making them suitable for a centralclinic with well-trained staff, while others are expensive, or notaccurate. Thus, there is a need for assay systems for detecting analytesthat are accurate, convenient, and inexpensive.

SUMMARY

The present invention provides methods and micro technological systemsfor performing assays for determining the presence of one or morepreselected analytes in a sample. The apparatus includes a disposableplastic strip that can be inserted into a portable handheld testanalysis machine. The strip isolates the sample such that it is not incontact with the machine and the sample is not contaminated.

The disposable strip of the present invention can have a plurality ofdefined wells on a solid support. The wells can be linked by capillarychannel. The surface of the wells and the capillary channels can becoated with reagents to assist in drawing the liquid sample from thesample well into the reaction wells. Inside at least one of the reactionchambers is at least one magnetic stir bar which can be attractedmagnetically and driven by a magnetic moving device arranged outside thestrip in the test analysis machine. The magnetic stir bar is capable ofexecuting motions that mix the reaction components, move reactioncomponents, exchange or systematically deliver reagents to targets inthe cartridge, and the like.

The strip can be placed on a portable handheld machine having sensorsconfigured to detect and or quantify the presence of an analyte in thereaction wells of the strip, while responding to the physicallydetectable changes, producing signals which correlate to the presence ofand or amount of the selected analyte in the sample. The reagents cancomprise the detection system, whereby a detectable result occurs inrelation to the presence of an analyte. The signal can be converted toan output on a visual display window on the external part of the base.

To test a sample, the disposable strip can be inserted into the base,and the sample drop can be placed in the sample application well of thestrip. The sample can be drawn into the reaction well through theinternal channels. As the sample is drawn into the reaction wells,sensors detect the movement and activate the magnetic stir bar at theappropriate times which mix the reagents within the sample well. Fortimed assays, a microprocessor contained within the base begins a timecount while the sensors, which can be electrical or optical, monitorvarious parts of the strip for specific analyte responses. When detectedand or quantified, the results are reported qualitatively orquantitatively with the appropriate units in the display window on thebase.

The motion and the sensors can be microprocessor controlled. A heaterassembly can be activated in the base, for temperature-sensitive assayssuch as coagulation tests and the temperature can be held constant orvaried in a predetermined way through the duration of the assay.

In one aspect of the invention, a disposable strip is described wherethe disposable strip comprises a first solid substrate comprising asample collection well, a reference well, and a reaction well, whereinthe wells are in fluidic communication via a first capillary channels;and a second solid substrate comprising holes wherein the first solidsubstrate and the second solid substrate are joined and the holescommunicate with the wells.

In another aspect of the invention, a disposable strip comprising afirst solid substrate comprising a sample application well, a referencewell, and a reaction well, wherein the wells are in fluidiccommunication via capillary channels, and wherein the reference wellcomprises a first lysing agent and the reaction well comprises a secondlysing agent, an antibody, and a stir bar; a second solid substratecomprising holes wherein the first solid substrate and the second solidsubstrate are joined and the holes communicate with the wells; and amembrane having a capture zone wherein the membrane is connected to thereaction well via a second capillary channel is described.

In yet another aspect, the invention pertains to a method fordetermining the percentage of hemoglobin that is HbA1c, the methodcomprising providing a disposable strip comprising a first solidsubstrate comprising a sample well, a reference well, and a reactionwell, wherein the wells are in fluidic communication via a firstcapillary channel, and wherein the reference well comprises a firstlysing agent and the reaction well comprises a second lysing agent, aHbA1c specific antibody, and a stir bar; a second solid substratecomprising holes wherein the first solid substrate and the second solidsubstrate are joined and the holes communicate with the wells; and amembrane having a capture zone wherein the membrane is connected to thereaction well via a second capillary channel; placing a sample in thesample application well; adding a diluent to the sample applicationwell; determining the total hemoglobin from the reference cell and thetotal HbA1c from the capture zone; and dividing the total HbA1c by totalhemoglobin to obtain the percentage of hemoglobin that is HbA1c.

These and other aspects of the present invention will become evidentupon reference to the following detailed description. In addition,various references are set forth herein which describe in more detailcertain procedures or compositions, and are therefore incorporated byreference in their entirety.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a perspective view of the disposable strip with a setof interconnected reaction chambers and a moveable member in one of thechambers.

FIG. 2 illustrates another view of the disposable strip comprising acomposite of membranes assembled to isolate the blood components anddirecting the desired analyte to the proper capture zones.

FIG. 3 illustrates a perspective view of the main components in the basedevice as described in detail below.

DETAILED DESCRIPTION

I. Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used herein, the term “subject” encompasses mammals and non-mammals.Examples of mammals include, but are not limited to, any member of theMammalian class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. The term does not denote a particular ageor gender.

The term “antibody,” as used herein, includes, but is not limited to apolypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof which specifically bind andrecognize an analyte (antigen). “Antibody” also includes, but is notlimited to, a polypeptide substantially encoded by an immunoglobulingene or immunoglobulin genes, or fragments thereof which specificallybind and recognize the antigen-specific binding region (idiotype) ofantibodies produced by the host in response to exposure to trichomonasantigen(s). Examples include polyclonal, monoclonal, chimeric,humanized, and single chain antibodies, and the like. Fragments ofimmunoglobulins, include Fab fragments and fragments produced by anexpression library, including phage display. See, e.g., Paul,Fundamental Immunology, 3^(rd) Ed., 1993, Raven Press, New York, forantibody structure and terminology.

The terms “specifically binds to” or “specifically immunoreactive with”refers to a binding reaction which is determinative of the presence ofthe target analyte in the presence of a heterogeneous population ofproteins and other biologics. Thus, under designated assay conditions,the specified binding moieties bind preferentially to a particulartarget analyte and do not bind in a significant amount to othercomponents present in a test sample. Specific binding to a targetanalyte under such conditions may require a binding moiety that isselected for its specificity for a particular target analyte. A varietyof immunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular antigen. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with an analyte. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity. Typically a specific orselective reaction will provide a signal to noise ratio at least twicebackground and more typically more than 10 to 100 times background.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, chromophores,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin,avidin, strepavidin or haptens) and the like.

As used herein, a “solid support” refers to a solid surface such as aplastic plate, magnetic bead, latex bead, microtiter plate well, glassplate, nylon, agarose, acrylamide, and the like.

“Specific” in reference to the binding of two molecules or a moleculeand a complex of molecules refers to the specific recognition of one forthe other and the formation of a stable complex as compared tosubstantially less recognition of other molecules and the lack offormation of stable complexes with such other molecules. Exemplary ofspecific binding are antibody-antigen interactions, enzyme-substrateinteractions, polynucleotide hybridizations and/or formation ofduplexes, cellular receptor-ligand interactions, and so forth.

II. Overview

The invention pertains to a disposable strip that can be used forperforming qualitative and quantitative immuno- and chemical assays. Onthe strip are at least three wells, where the wells can be in fluidiccommunication with each other via capillary channels. In one well isplaced the sample, preferably a liquid sample, for analysis. The samplefluid moves into the other two wells via the capillary channels. One ofthe wells can serve as a standard that measures the total analyte in thesample. The other well can serve as the reaction well, where individualcomponents of the sample can be identified. The disposable strip can beplaced in an analyzer (also referred to herein as a “base”) that detectsthe individual components of the sample and the total analyte in thesample. The analyzer includes a display system that can display theresults of the analysis as well as provided instructions during theoperation of the assay.

In one application, the percent total hemoglobin that is hemoglobin A1c(HbA1c) in human red blood cell can be determined. Blood from a subjectcan be deposited in the sample well. The blood is moved into the othertwo wells via the capillary channels. In the reference well can beplaced a reagent that lyses the cells thereby releasing the hemoglobinfrom the red blood cells. The concentration of hemoglobin in thereference cell can be measured using infrared or ultravioletmeasurements. In the reaction well can be placed a lysate, a knownamount of an antibody specific for HbA1c, and a magnetic stirrer. Whenblood moves into the reaction well, the magnet stirs the liquids in thewell thereby mixing them well. The lysate lyses the cells, and theantibody binds to HbA1c. After a specified period of time, the displaycan instruct the operator to add a diluent to the reaction well. Thediluent pushes the liquid in the reaction chamber through anothercapillary channel towards one or more capture zones. The capture zoneshave immobilized on them antigens that bind to the bound antibodycomplex only, and on a separate part of the zone other antigens thatbind to all antibodies. The antibody-HbA1c complex can be captured bythe antigens in the first part of the capture zone, and all theantibodies can be captured by the antigens in the latter part of thecapture zone. A detection system can be used to detect the antibodiesbound in the first and the second part of the capture zone. The ratioand/or the sum of the two zones can be used to quantify the amount ofHbA1c present in the sample. The ratio of the first zone to the totalhemoglobin from the reference cell can provide the percentage of HbA1cin the blood sample. The results can be displayed on the display system

III. Micro Mechanical System

The invention provides a disposable strip, a portable handheld machine(i.e., a base), and a combination comprising a disposable strip and abase. The strip can be placed on the machine for performing assays, fordetecting analytes, and for displaying information such as instructionsand results. One aspect of the disposable strip is illustrated inFIG. 1. The disposable strip can be made by joining together two or moresolid supports with grooves present in at least one of the supports. Thesolid support can be rectangular, circular, oval, or any shape. Thesupport can be made from a suitable material that is selected on itsproperties, such as good thermal conductivity, clarity for opticaltransmission, mechanical properties for easy welding, surface propertiesthat allow for uniform coating and stability of reagent, and neutralityto the liquid medium to prevent interference with the assay. For thispurpose, suitable plastics include those with high free surface energiesand low water sorption, including PETG, polyester (Mylar®),polycarbonate (Lexan®), polyvinyl chloride, polystyrene, SAN,acrylonitrile-butadiene-styrene (ABS), particularly ABS supplied by BorgWarner under the trade name Cycolac, among others. When the solidsupport is a hydrophobic plastic, it can be treated by art-known methodsto render the surfaces hydrophilic, such as by plasma etching and bycorona treatment. Alternatively and equivalently, acommercially-available molded solid support can be used in the practiceof the invention.

For purposes of illustration, this embodiment of the invention isdescribed by reference to a disposable strip formed by joining two solidsupports. At least one of the solid supports has grooves or cavitiesthat serve as the reaction chambers 5 and 7, and capillary channels 2and 8. The grooves can be any geometric shape, and are preferablycircular. The grooves have dimensions that are sufficient volume to holdthe samples and to allow for the reaction to occur. Thus, the circulargrooves can have a diameter of between about 0.01 mm to about 100 mm,depending on the length and width of the support material, and can havea height of about 0.001 mm to about 4 mm, depending on the thickness ofthe support material. The diameter and height of the grooves can beeasily determined by the one of skill in the art. In one aspect of theinvention, one of the support pieces has holes drilled through to thegrooves where the holes serve as the vent holes 3 and 6. Further, theholes can allow access to the well where the sample will be placed, suchas the sample application well, 1. Prior to the joining of the twopieces, the moveable member, 4, can be inserted in the desired reactionchamber, 5.

In the molding process, energy-directing ridges are needed at least onthe outline adjacent to the periphery of the groove of at least one ofthe two plastic pieces. When welded ultrasonically, the two plasticpieces are glued together along the energy ridges forming an air-tightseal around the chambers and channels, with the only access to theexterior from the reaction chambers being the vent holes and sampleapplication well. The surface of the reaction chamber can optionally beslightly textured for use with moveable members. The texturing canaccommodate a disjoining pressure, Π. Π, is the pressure, in the case oftwo plates immersed in a medium, in excess of the external pressure,that must be applied to the medium between the plates to maintain agiven separation. In this case, Π is numerically just the force ofattraction or repulsion between the moveable member and the surfaces ofthe reaction chamber per unit area. The wider the moveable member, thegreater would be the pressure between the surfaces and texturing wouldeliminate any undesired clamping of the moveable member unto the wallsof the reaction chamber. A more general definition for disjoiningpressure isΠ=−1/{haeck over (A)}({haeck over (o)}G/{haeck over (o)}x) {haeck over(A)}, T, V where

-   -   {haeck over (A)}=Area    -   T=Temperature    -   V=Volume    -   G=Gibb's free energy

The moveable member, 4, can be made by use of stainless steel or acombination of stainless steel with any other desired material so thatit is capable of being attracted and driven by an external magneticmoving device. The material can be any form of magnetizable alloy with astainless covering to prevent corrosion or specially coated for bondingof specific molecules. The thickness of the movable member is based onthe height of the reaction chamber. It has to be small enough to fit inthe reaction chamber and move freely. For a reaction chamber cavity of aheight of 0.010 inches, the thickness of the moveable member can bebetween about 0.007 to about 0.008 inches.

The mode for applying the sample to the reaction chambers as well asother reagents, such as salt and sugar solutions to the capillarychannels include spraying, painting, lyophilization, evaporation,adsorption, covalent conjugation or the like. For reagents with largeparticulate components, spray painting or lyophilization would beadequate. Biodeposition of pico liter drop sizes results ininstantaneous drying when dispensed at room temperature due to the sizeof the drops.

An assembled disposable strip is illustrated in FIG. 2, where 5 is anabsorbent pad, 7 is an internal reference membrane strip, 9 is acapillary channel, 11 is a reaction chamber, 13 is a capillary channel,15 is a sample receptacle, 17 is a sample application port, 19 is areaction chamber, 21 is a moveable member, 23 are capture zones on abase membrane 25.

FIG. 3 is a perspective diagram of the main components of the analyzerinto which the disposable strip is inserted prior to applying a testsample. The disposable strip can be positioned at 46 on the base. Thestrip can be placed on top of a heater assembly 44 that may accommodatea sensor (emitters and or detectors) embedded in it or in closeproximity but arranged such that a signal goes through the reactionchamber. Another set of sensors that also can serve as emitters,detectors, or both can be positioned on the other side of the disposablestrip (not shown). It is to be understood that the reflective beamarrangement, the detector and the emitter can be on the same side of thestrip, depending on the detection mechanism that is used. The detectionmechanism is not limited to an optical detection method, but othermethods, such as electrical, radioactive, and other methods could alsobe used. An electronic display window 30 can be, but is not limited to,a Liquid Crystal Display, LCD. 28, represents switches or keys on amembrane for function selection from a menu displayed on the LCD. Theelectronic board 36 comprises a microprocessor that controls theoperation and mechanics of the analyzer. The power can be supplied bybatteries 38, or any other source of alternate electrical power supply.The motor 40 drives the magnet, 42, and the two can be connected, suchas by a rod. The defined motion pattern of the motor and magnet areresponsible for creating the corresponding motion of the moveable memberin one or more of the reaction chambers of the disposable strip. 32,represents a connector to an external power supply and 34 represents aconnector for data output.

An example of a detection system for automated detection for use withthe present disposable strip and associated methods comprises anexcitation source, a monochromator (or any device capable of spectrallyresolving light components, or a set of narrow band filters) and adetector array. The excitation source can comprise infrared, blue or UVwavelengths and the excitation wavelength can be shorter than theemission wavelength(s) to be detected. The detection system may be: abroadband UV light source, such as a deuterium lamp with a filter infront; the output of a white light source such as a xenon lamp or adeuterium lamp after passing through a monochromator to extract out thedesired wavelengths; or any of a number of continuous wave (cw) gaslasers, including but not limited to any of the Argon Ion laser lines(457, 488, 514, etc. nm) or a HeCd laser; solid-state diode lasers inthe blue such as GaN and GaAs (doubled) based lasers or the doubled ortripled output of YAG or YLF based lasers; or any of the pulsed laserswith output in the blue.

The emitted light from the sample or the reactants in the reaction wellcan be detected with a device that provides spectral information for thesubstrate, e.g., a grating spectrometer, prism spectrometer, imagingspectrometer, or the like, or use of interference (bandpass) filters.Using a two-dimensional area imager such as a CCD camera, many objectsmay be imaged simultaneously. Spectral information can be generated bycollecting more than one image via different bandpass, longpass, orshortpass filters (interference filters, or electronically tunablefilters are appropriate). More than one imager may be used to gatherdata simultaneously through dedicated filters, or the filter may bechanged in front of a single imager. Imaging based systems, like theBiometric Imaging system, scan a surface to find fluorescent signals.

Other embodiments appropriate for this system include the use ofreagent-coated membranes systems as part of the strip positioned in away that allows continuity and directed sample flow within the entirestrip system. The sensory systems would be positioned to be capable ofmonitoring the membrane portions of the strip for the analyte orresponses being tested for.

IV. Operation

A general mode of operation of the device shown in FIG. 3 involves theinsertion of the disposable strip shown in FIGS. 1 and 2 into areceptacle that allows the strip on one position only. For an assay witha specific temperature requirement, the heater assembly, 44, heats thedisposable strip to the desired temperature controlled by themicroprocessor. The LCD prompts simple steps, after the strip isinserted and the analyzer turned on, which the operator can followincluding the addition of the sample to the sample receptacle. Theinstrument can optionally have sensors to determine the presence ofadequate amounts of sample in the reaction chambers and a mechanism toinitiate and stop the timing of the assay. The sensor detect the signalsfrom the completion of the reaction, such as measuring the transmissionof an optical signal emitted and directed through the walls of thereaction chamber.

The applied sample is accurately distributed into the various reactionchambers via the capillary channels. The positioning of the reactionchambers can be such that independent reactions can occur in the variousreaction chambers even though they share a common sample from the samepool. The defined modes of movement of the moveable member ensuresproper mixing of the reagent and sample mixtures and also contributes ininter chamber reagent and sample interchange. For assays that requirequantification of an analyte, the sensory system monitors the changeseither in one or more reaction chambers, a membrane system or themoveable members, until the desired end point is achieved. For assaysrequiring just the determination of the presence of an analyte, thesensory system monitors the specific parts of the strip for theappropriate duration of time. The microprocessor computes the resultsquantitatively or qualitatively, which are displayed on the LCD. Thestrip can then be removed at the end of the assay and disposed.

Thus, the operator inserts the strip into strip receptacle in theanalyzer. The operator then pushes a start button, which couldautomatically be activated by the strip itself, waits for a prompt toadd a drop of sample, and then obtains the results from the display,typically within a few minutes or seconds, depending on the assay type.

V. Detection of Hemoglobin A1c (HbA1c)

Glycated hemoglobin refers to a series of minor hemoglobin componentsthat are formed through the attachment of glucose to the hemoglobinmolecule. The human red blood cell is freely permeable to glucose.Within each red blood cell, glycated hemoglobin is formed at a rate thatis directly proportional to the ambient glucose concentration.Approximately 97% of the total hemoglobin in circulating red blood cellsis hemoglobin A. Hemoglobin A consists of four polypeptide chains, twoa-chains and two b-chains. Glycation of the Hemoglobin A occurs throughthe covalent coupling of glucose with the N-terminal valine amino acidof each b-peptide chain. An unstable Schiff base (aldimine) is initiallyformed which then undergoes an irreversible Amadori rearrangement toform a stable ketoamine, Hemoglobin A1c (HbA1c).

The life-span of hemoglobin A containing red blood cells averages 120days. The percentage of Hemoglobin A that is glycated to HbA1c isdirectly proportional to the time that red blood cells are exposed toglucose and to the average glucose concentration encountered.Measurement of the HbA1c fraction gives an integrated picture of theaverage blood glucose concentration during the half-life of the redcells, that is, over the last 60 days. The level of HbA1c is usuallyexpressed as a percentage of total hemoglobin.

In normal subjects, HbA1c is typically in the range 3-6% of totalhemoglobin. In patients with elevated glucose levels e.g. in the case ofType I and Type 2 diabetes, the level may rise to twice the upper limitof normal or more.

Long-term control of glucose levels in diabetics is very important. Toomuch glucose in the blood over many years can damage the eyes, kidneysand nerves. It also increases the risk for heart and blood vesseldisease. The measurement of HbA1c as a percentage of total hemoglobinprovides a valuable means of assessing the long-term control of glucoselevels and also constitutes an important risk indicator for identifyingType 1 and Type 2 diabetics.

A sample of blood from a subject can be obtained in deposited in thesample well 15 of the disposable strip (FIG. 2). The blood moves to thereaction chambers 11 and 19 via the capillary channels 13. The reactionchamber 11 can serve as the reference where the total hemoglobin ismeasured. The reaction chamber 19 measures the HbA1c in the bloodsample. The ratio of HbA1c to the total hemoglobin provides thepercentage of total hemoglobin that is HbA1c.

Both of the reaction chambers contain a lysing agent. The lysing agentlyses the whole blood samples thereby releasing the hemoglobin. Thelysing agents are typically surfactants, and preferably nonionicsurfactants, such as for example TRITON™ X-100. The reaction chamber 19additionally contains an antibody that can detect HbA1c. The antibodycan be a monoclonal or polyclonal antibody (Ab), or Ab fragmentcontaining the antigen binding site, or complementarity determiningregion (CDR), such as an F(ab′)₂ or Fab fragment. The detectable moietyor label may be a radioactive, fluorescent or chemiluminescentsubstance, or an enzyme. Alternatively, a labeled-second Ab whichrecognizes the species specific Fc fragment of the first Ab may also beused. Further, the antibody may be labeled with a detectable label.

In one aspect, the detectable label is a fluorescent molecule. Examplesof suitable fluorescent labels include fluorescein (FITC),5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,rhodamine, 4′-6-diamidino-2-phenylinodole (DAPI), and the cyanine dyesCy3, Cy3.5, Cy5, Cy5.5 and Cy7. Preferred fluorescent labels arefluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine(5,6-tetramethyl rhodamine), substituted rhodamine compounds, and thecyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. The absorption and emissionmaxima, respectively, for these fluorophores are: FITC (490 nm; 520 nm),Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm),Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm), thus allowing theirperoxidase-like properties, can generate a detectable signal. This isaccomplished by adding hydrogen peroxide, with or without addition ofanother substrate (e.g. isoluminol).

The cells in the two reaction wells are lysed. In the reference well 11,the total amount of hemoglobin can be obtained by spectroscopic methods,such as measuring in the UV region or the infrared region. Thespectroscopic apparatus is known in the art and is incorporated withinthe portable handheld machine. In particular, the measurements can bemade at 880 nm and at 580 nm. In the reaction well 19, the antibodiesbind to HbA1c. In order to ensure complete reaction, the liquids in thereaction wells can be magnetically stirred, and optionally heated to ahigher temperature.

Upon completion of the reaction, a diluent can be added to the samplewell 15. The diluent causes the reactants in 19 to move through thecapillary channels 9 towards the capture zones 23. The capture zones canbe antigens or other compounds that can specifically bind to theantibody-HbA1c complex, any antibody, and the like or combinationsthereof. Thus, in one aspect, the first capture zone contains antigensthat specifically bind to the antibody-HbA1c complex, while the secondcapture zone contains antigens that bind to the antibody and theantibody-HbA1c complex. The absorbent pad 5 absorbs all the liquid andcan help in drawing the liquid from the wells through the membranes.

The amount of material in each capture zone can be determined by usingthe detection systems described above. Calibrators or standards that arerun with the assay provide calibration (or standard) curves from whichthe % HbA1c in the sample is determined using the measured signal. Thesum of all the capture zones preferably equals the amount of antibodythat was placed in the reaction well, and can provide an internalcontrol to determining the percentage of reaction that has occurred. Theconcentration of the antibody-HbA1c complex can be determined from thereading of the first capture zone. The % HbA1c in the blood sample canbe determined by dividing the concentration of the antibody-HbA1ccomplex with the total concentration of hemoglobin.

In another aspect, a reference membrane 7 (FIG. 2) can be included inthe disposable strip. The reference membrane can have deposited upon itknown concentrations of the antigen-antibody-HbA1c complex, andantigen-antibody complex. The spectrophotometric measurements form thereference membrane can be used to calibrate the readings from the activemembrane 25.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention. All printedpatents and publications referred to in this application are herebyincorporated herein in their entirety by this reference.

1. A disposable strip comprising: a first solid substrate comprising a sample collection well, a reference well, and a reaction well, wherein the wells are in fluidic communication via a first capillary channels; and a second solid substrate comprising holes wherein the first solid substrate and the second solid substrate are joined and the holes communicate with the wells.
 2. The disposable strip of claim 1, wherein the first substrate and the second substrate are rectangular.
 3. The disposable strip of claim 1, wherein the substrate is selected from the group consisting of plastic, glass, nylon, metal, and combinations thereof.
 4. The disposable strip of claim 3, wherein the substrate is plastic.
 5. The disposable strip of claim 1, further comprising a stir bar in the reaction well.
 6. The disposable strip of claim 1, further comprising an antibody deposited in the reaction well.
 7. The disposable strip of claim 6, wherein the antibody is specific for HbA1c.
 8. The disposable strip of claim 1, wherein the reference well and the reaction well comprise a lysing agent.
 9. The disposable strip of claim 1, wherein the lysing agent is a nonionic surfactant.
 10. The disposable strip of claim 1, further comprising a membrane having a capture zone wherein the membrane is connected to the reaction well via a second capillary channel.
 11. The disposable strip of claim 10, wherein the membrane is interposed between an absorbent pad and the reaction well.
 12. A disposable strip comprising: a first solid substrate comprising a sample application well, a reference well, and a reaction well, wherein the wells are in fluidic communication via capillary channels, and wherein the reference well comprises a first lysing agent and the reaction well comprises a second lysing agent, an antibody, and a stir bar; a second solid substrate comprising holes wherein the first solid substrate and the second solid substrate are joined and the holes communicate with the wells; and a membrane having a capture zone wherein the membrane is connected to the reaction well via a second capillary channel.
 13. The disposable strip of claim 12, wherein the first substrate and the second substrate are rectangular.
 14. The disposable strip of claim 1, wherein the substrate is selected from the group consisting of plastic, glass, nylon, metal, and combinations thereof.
 15. The disposable strip of claim 14, wherein the substrate is plastic.
 16. The disposable strip of claim 12, wherein the first and the second lysing agents are nonionic surfactant.
 17. The disposable strip of claim 12, wherein the antibody is specific for HbA1c.
 18. A method for determining the percentage of hemoglobin that is HbA1c, the method comprising: providing a disposable strip comprising a first solid substrate comprising a sample well, a reference well, and a reaction well, wherein the wells are in fluidic communication via a first capillary channel, and wherein the reference well comprises a first lysing agent and the reaction well comprises a second lysing agent, a HbA1c specific antibody, and a stir bar; a second solid substrate comprising holes wherein the first solid substrate and the second solid substrate are joined and the holes communicate with the wells; and a membrane having a capture zone wherein the membrane is connected to the reaction well via a second capillary channel; placing a sample in the sample application well; adding a diluent to the sample application well; determining the total hemoglobin from the reference cell and the total HbA1c from the capture zone; and dividing the total HbA1c by total hemoglobin to obtain the percentage of hemoglobin that is HbA1c.
 19. The method of claim 18, wherein the first substrate and the second substrate are rectangular.
 20. The method of claim 18, wherein the substrate is selected from the group consisting of plastic, glass, nylon, metal, and combinations thereof.
 21. The method of claim 20, wherein the substrate is plastic.
 22. The method of claim 18, wherein the first and second lysing agents are nonionic surfactant.
 23. The method of claim 18, wherein the membrane comprises two or more capture zones.
 24. The method of claim 23, wherein one capture zone comprises antigens that specifically bind to antibody-HbA1c complex, and another capture zone comprises antigens that bind to the antibody.
 25. The method of claim 24, wherein the total HbA1c is determined by UV measurements.
 26. The method of claim 18, wherein the total hemoglobin is determined by UV, IR or combinations thereof.
 27. The method of claim 18, wherein the total hemoglobin is spectrophotometrically determined using 580 nm and 880 nm light. 